Colocalization of Sensors Is Sufficient to Activate the DNA Damage Checkpoint in the Absence of Damage  Carla Yaneth Bonilla, Justine Amy Melo, David Paul Toczyski  Molecular Cell  Volume 30, Issue 3, Pages 267-276 (May 2008) DOI: 10.1016/j.molcel.2008.03.023 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Colocalization of Checkpoint Fusions Promotes Rad53 and Rad9 Phosphorylation (A) Inducible checkpoint fusions' experimental design. (B) Colocalization of Ddc1 and Ddc2 at a LacO array does not cause Rad52 foci formation. Cells containing Ddc1-GFP-LacI, Ddc2-GFP-LacI, LacO array, and Rad52-RFP were arrested with nocodazole and treated as follows: 100 ug/mL Zeocin for 3 hr in rich media plus dextrose, rich media plus dextrose, and rich media plus a 1 hr galactose pulse. Digital microscopy was used to measure the frequency of Rad52-RFP foci formation in all samples. (C–E) Strains were arrested with nocodazole and maintained arrested while galactose was added to induce fusions or the HO endonuclease as a positive control. Rad53-HA (C and D) and Rad9-HA were visualized with anti-HA antibody (E). Checkpoint fusions were visualized with anti-GFP antibodies. (F) Arrays of tandem LacO repeats were introduced in the strain coexpressing the checkpoint fusions. Strains were nocodazole arrested (top panel), and galactose was added to induce fusion expression for 2.5 hr (bottom panel). Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Colocalization Activates Multiple Checkpoint Readouts (A) Sml1 is degraded upon colocalization. A wild-type strain was left untreated or was treated 100 ug/ml Zeocin for 4 hr. Strains expressing checkpoint fusions −array and +array were arrested with nocodazole, and galactose was added to induce fusions. The westerns were blotted against endogenous Sml1 and Rad53-HA. A sml1 delete strain served as control for specificity (last lane). (B–D) Strains containing fusions −array (B), +array (C), and +array, rad9 (D) were arrested in nocodazole, induced with galactose for 1 hr while arrested, and then released into media with α factor. A time course of FACS analysis is shown starting at 2 hr (120′) into galactose induction. (E) Analysis of Rad53-HA by western blot of samples taken from (B)–(D). Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 9-1-1 and Ddc2-Mec1 Colocalization Bypasses the Requirement for Rad24 (A) Both checkpoint fusions were induced in nocodazole-arrested isogenic wild-type, rad24, rad9, and mec1 strains containing a LacO array. (B) Checkpoint fusions were induced in nocodazole-arrested wild-type and rad24 strains. Galactose and Zeocin were added simultaneously in the presence of nocodazole. Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Ddc1 Is the Mec1-Activating Subunit (A) Ddc1-GFP-LacI and Ddc2-GFP-LacI checkpoint fusions were induced in nocodazole-arrested isogenic wild-type, mec3, and rad17 strains containing a LacO array. (B) Mec3-GFP-LacI and Ddc2-GFP-LacI checkpoint fusions were induced in nocodazole-arrested isogenic wild-type and ddc1 strains containing a LacO array. Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Rad53 Activation Requires H2AX-P and H3K79Me (A) Ddc1-GFP-LacI and Ddc2-GFP-LacI checkpoint fusions were induced in nocodazole-arrested isogenic wild-type, htaS129; dot1, and htaS129, dot1 strains containing a LacO array. (B) Same as in (A) but for a longer time. Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 CDK Promotes Rad53 Activation (A and B) Strains were arrested in G1 with α factor and G2/M with nocodazole, and galactose was added to induce checkpoint fusions. (C) Checkpoint fusions were induced in nocodazole-arrested isogenic wild-type and analog (1NM-PP1)-sensitive, cdc28-as, strains containing a LacO array. Both strains were treated as follows: inhibitor alone (5 uM), galactose plus inhibitor, and galactose alone. (D) The strain carrying the cdc28-as allele, checkpoint fusions, and array was arrested with nocodazole and induced with galactose. After 3 hr, the culture was split and inhibitor was added. Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Rad9 CDK Site Mutant Is Checkpoint Deficient (A) Isogenic strains carrying wild-type Rad9-Flag or Rad9-18A-Flag were α factor arrested, nodocazole arrested, allowed to cycle, or treated with cdc28-as inhibitor 1NM-PP1. (B) Ddc1-GFP-LacI and Ddc2-GFP-LacI checkpoint fusions were induced in nocodazole-arrested Rad9-Flag and Rad9-18A-Flag strains. Zeocin was added to Rad9-Flag, Rad9-18A-Flag, and rad9Δ strains. (C) Rad9-Flag, Rad9-18A-Flag, and rad9Δ cycling strains were left untreated or treated with Zeocin at 100 ug/ml, 200 ug/ml, and 2 mg/ml final concentration for 2 hr. Molecular Cell 2008 30, 267-276DOI: (10.1016/j.molcel.2008.03.023) Copyright © 2008 Elsevier Inc. Terms and Conditions